Calcium channel blockers comprising two benzhydril moieties

ABSTRACT

Certain piperazine substituted compounds are described which are useful in altering calcium channel activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/409,763 filed 8Apr. 2003 and now pending, which is a continuation-in-part of U.S. Ser.No. 10/060,900 filed 29 Jan. 2002, now U.S. Pat. No. 6,617,322, which isa continuation of U.S. Ser. No. 09/476,927 filed 30 Dec. 1999, now U.S.Pat. No. 6,387,897; which is a continuation-in-part of U.S. Ser. No.09/401,699, filed 23 Sep. 1999, now U.S. Pat. No. 6,294,533; which is acontinuation-in-part of U.S. Ser. No. 09/107,037 filed 30 Jun. 1998, nowU.S. Pat. No. 6,011,035. The contents of these applications areincorporated herein by reference.

TECHNICAL FIELD

The invention relates to compounds useful in treating conditionsassociated with abnormal calcium channel function. More specifically,the invention concerns compounds containing substituted or unsubstitutedderivatives of 6-membered heterocyclic moieties that are useful intreatment of conditions such as stroke and pain.

BACKGROUND ART

PCT publication WO 01/45709 published 28 Jun. 2001 discloses calciumchannel blockers where a piperidine or piperazine ring links abenzhydril moiety to an additional aromatic moiety or benzhydril. Thispublication, which is based on parent application Ser. No. 09/476,927,discussed above, is incorporated herein by reference. As explained inthese applications, native calcium channels have been classified bytheir electrophysiological and pharmacological properties as T, L, N, Pand Q types. T-type (or low voltage-activated) channels describe a broadclass of molecules that transiently activate at negative potentials andare highly sensitive to changes in resting potential. The L, N, P andQ-type channels activate at more positive potentials (high voltageactivated) and display diverse kinetics and voltage dependentproperties. There is some overlap in biophysical properties of the highvoltage activated channels, consequently pharmacological profiles areuseful to further distinguish them. Whether the Q- and P-type channelsare distinct molecular entities is controversial. Several types ofcalcium conductances do not fall neatly into any of the above categoriesand there is variability of properties even within a category suggestingthat additional calcium channels subtypes remain to be classified.

Biochemical analyses show that neuronal high voltage activated calciumchannels are heterooligomeric complexes consisting of three distinctsubunits (α₁, α₂δ and β). The α₁ subunit is the major pore-formingsubunit and contains the voltage sensor and binding sites for calciumchannel antagonists. The mainly extracellular α₂ is disulfide-linked tothe transmembrane δ subunit and both are derived from the same gene andare proteolytically cleaved in vivo. The β subunit is a nonglycosylated,hydrophilic protein with a high affinity of binding to a cytoplasmicregion of the α₁ subunit. A fourth subunit, γ, is unique to L-typecalcium channels expressed in skeletal muscle T-tubules.

Recently, each of these α₁ subtypes has been cloned and expressed, thuspermitting more extensive pharmacological studies. These channels havebeen designated α_(1A)-α_(1I) and α_(1S) and correlated with thesubtypes set forth above. α_(1A) channels are of the P/Q type; α_(1B)represents N; α_(1C), α′_(1D), α_(1F) and α_(1S) represent L; α_(1E)represents a novel type of calcium conductance, and α_(1G)-α_(1I)represent members of the T-type family.

Further details concerning the function of N-type channels, which aremainly localized to neurons, have been disclosed, for example, in U.S.Pat. No. 5,623,051, the disclosure of which is incorporated herein byreference. As described, N-type channels possess a site for bindingsyntaxin, a protein anchored in the presynaptic membrane. Blocking thisinteraction also blocks the presynaptic response to calcium influx.Thus, compounds that block the interaction between syntaxin and thisbinding site would be useful in neural protection and analgesia. Suchcompounds have the added advantage of enhanced specificity forpresynaptic calcium channel effects.

U.S. Pat. No. 5,646,149 describes calcium channel antagonists of theformula A-Y-B wherein B contains a piperazine or piperidine ringdirectly linked to Y. An essential component of these molecules isrepresented by A, which must be an antioxidant; the piperazine orpiperidine itself is said to be important. The exemplified compoundscontain a benzhydril substituent, based on known calcium channelblockers (see below). In some cases, the antioxidant can be a phenylgroup containing methoxy and/or hydroxyl substituents. In most of theillustrative compounds, however, a benzhydril moiety is coupled to theheterocycle simply through a CH group or C═ group. In the few compoundswhere there is an alkylene chain between the CH to which the two phenylgroups are bound and the heterocycle, the antioxidant must be coupled tothe heterocycle through an unsubstituted alkylene and in most of thesecases the antioxidant is a bicyclic system. Where the antioxidant cansimply be a phenyl moiety coupled through an alkynylene, the linker fromthe heterocycle to the phenyl moieties contains no more than six atomsin the chain. U.S. Pat. No. 5,703,071 discloses compounds said to beuseful in treating ischemic diseases. A mandatory portion of themolecule is a tropolone residue; among the substituents permitted arepiperazine derivatives, including their benzhydril derivatives. U.S.Pat. No. 5,428,038 discloses compounds which are said to exert a neuralprotective and antiallergic effect. These compounds are coumarinderivatives which may include derivatives of piperazine and othersix-membered heterocycles. A permitted substituent on the heterocycle isdiphenylhydroxymethyl. Thus, approaches in the art for variousindications which may involve calcium channel blocking activity haveemployed compounds which incidentally contain piperidine or piperazinemoieties substituted with benzhydril but mandate additional substituentsto maintain functionality.

Certain compounds containing both benzhydril moieties and piperidine orpiperazine are known to be calcium channel antagonists and neurolepticdrugs. For example, Gould, R. J., et al., Proc Natl Acad Sci USA (1983)80:5122-5125 describes antischizophrenic neuroleptic drugs such aslidoflazine, fluspirilene, pimozide, clopimozide, and penfluridol. Ithas also been shown that fluspirilene binds to sites on L-type calciumchannels (King, V. K. et al., J Biol Chem (1989) 264:5633-5641) as wellas blocking N-type calcium current (Grantham, C. J., et al., Brit JPharmacol (1944) 111:483-488). In addition, Lomerizine, as developed byKanebo K K, is a known calcium channel blocker; Lomerizine is, however,not specific for N-type channels. A review of publications concerningLomerizine is found in Dooley, D., Current Opinion in CPNSInvestigational Drugs (1999) 1:116-125.

In addition, benzhydril derivatives of piperidine and piperazine aredescribed in PCT publication WO 00/01375 published 13 Jan. 2000 andincorporated herein by reference. This PCT publication corresponds toparent application 09/401,699 set forth above. Reference to this type ofcompound as known in the prior art is also made in WO 06/18402 published6 Apr. 2000 and in Chiarini, A., et al., Bioorganic and MedicinalChemistry, (1996) 4:1629-1635.

Various other piperidine or piperazine derivatives containing arylsubstituents linked through nonaromatic linkers are described as calciumchannel blockers in U.S. Pat. No. 5,292,726; WO 99/43658; Breitenbucher,J. G., et al., Tet Lett (1998) 39:1295-1298.

The present invention is based on the recognition that the combinationof a six-membered heterocyclic ring containing at least one nitrogensaid nitrogen coupled through a linker to a benzhydril moiety results ineffective calcium channel blocking activity. In some cases enhancedspecificity for N-type and/or T-type channels, or decreased specificityfor L-type channels is shown. The compounds are useful for treatingstroke and pain and other calcium channel-associated disorders, asfurther described below. By focusing on these moieties, compounds usefulin treating indications associated with calcium channel activity areprepared.

DISCLOSURE OF THE INVENTION

The invention relates to compounds useful in treating conditions such asstroke, head trauma, migraine, chronic, neuropathic and acute pain,epilepsy, hypertension, cardiac arrhythmias, and other indicationsassociated with calcium metabolism, including synaptic calciumchannel-mediated functions. The compounds of the invention arebenzhydril derivatives of piperazine with substituents that enhance thecalcium channel blocking activity of the compounds. Thus, in one aspect,the invention is directed to compounds of the formula

wherein each R¹-R⁵ is independently optionally substituted alkyl(1-10C), alkenyl (2-10C), alkynyl (2-10C), aryl (6-10C), arylalkyl(7-16C) or arylalkenyl (7-16C) each optionally further containing 1-4heteroatoms (N, O or S) and wherein said optional substituents mayinclude ═O thus including embodiments wherein R¹-R⁵ may independentlyform an acyl, amide, or ester linkage with the ring carbon to which itis bound, or each of R¹-R⁵ is independently halo, CF₃, OCF, NO₂, NR₂,OR, SR, COOR, or CONR₂, wherein R is H or optionally substituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, or arylalkenyl, as described above,and wherein two substituents at adjacent positions on the same ring mayform a 3-7 membered saturated or unsaturated ring fused to saidsubstituted ring, said fused ring optionally itself substituted andoptionally containing one or more heteroatoms (N, S, O), and R³ may beketo if n³=1;

-   -   or wherein a combination of R¹ and R² and/or R⁴ and R⁵ may form        a bond or a bridge between phenyl groups A and B and/or C and D        -e.g., each of R¹ and R² or R⁴ and R⁵ together may be a bond or        a single CR₂ group, an NR group, an O, or S wherein the S is        optionally oxidized; and

wherein each n¹-n⁵ is independently 0-4.

The invention is also directed to methods to modulate calcium channelactivity, preferably N-type and/or T-type channel activity, using thecompounds of formula (1) and thus to treat certain undesirablephysiological conditions; these conditions are associated with abnormalcalcium channel activity. In another aspect, the invention is directedto pharmaceutical compositions containing these compounds, and to theuse of these compounds for the preparation of medicaments for thetreatment of conditions requiring modulation of calcium channelactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows illustrative compounds of the invention.

FIG. 2 is a graph showing the selectivity of compound P1 for N-, P/Q-and L-type channels.

FIG. 3 is a graph showing the selectivity of compound P3 for N-, P/Q-and L-type channels.

FIG. 4 is a graph showing the selectivity of compound P4 for N-, P/Q-and L-type channels.

FIG. 5 is a graph showing the selectivity of compound P5 for N-, P/Q-and L-type channels.

FIG. 6 is a graph showing the selectivity of compound P6 for N-, P/Q-and L-type channels.

FIG. 7 is a graph showing the selectivity of compound P8 for N-, P/Q-and L-type channels.

MODES OF CARRYING OUT THE INVENTION

The compounds of formula (1) useful in the methods of the inventionexert their desirable effects through their ability to modulate theactivity of N-type and/or T-type calcium channels. This makes themuseful for treatment of certain conditions. Among such conditions whereantagonist activity is desired are stroke, epilepsy, head trauma,migraine, inflammatory bowel disease and chronic, neuropathic and acutepain. Calcium flux is also implicated in other neurological disorderssuch as schizophrenia, anxiety, depression, other psychoses, and neuraldegenerative disorders. Other treatable conditions includecardiovascular conditions such as hypertension and cardiac arrhythmias.In addition, T-type calcium channels have been implicated in certaintypes of cancer, diabetes, infertility and sexual dysfunction.

While the compounds of formula (1) generally have this activity,availability of this class of calcium channel modulators permits anuanced selection of compounds for particular disorders. Theavailability of this class of compounds provides not only a genus ofgeneral utility in indications that are affected by excessive calciumchannel activity, but also provides a large number of compounds whichcan be mined and manipulated for specific interaction with particularforms of calcium channels. The availability of recombinantly producedcalcium channels of the α_(1A)-α_(1I) and α_(1S) types set forth above,facilitates this selection process. Dubel, S. J., et al., Proc Natl AcadSci USA (1992) 89:5058-5062; Fujita, Y., et al., Neuron (1993)10:585-598; Mikami, A., et al., Nature (1989) 340:230-233; Mori, Y., etal., Nature (1991) 350:398-402; Snutch, T. P., et al., Neuron (1991)7:45-57; Soong, T. W., et al., Science (1993) 260:1133-1136; Tomlinson,W. J., et al., Neuropharmacology (1993) 32:1117-1126; Williams, M. E.,et al., Neuron (1992) 8:71-84; Williams, M. E., et al., Science (1992)257:389-395; Perez-Reyes, et al., Nature (1998) 391:896-900; Cribbs, L.L., et al., Circulation Research (1998) 83:103-109; Lee, J. H., et al.,Journal of Neuroscience (1999) 19:1912-1921.

It is known that calcium channel activity is involved in a multiplicityof disorders, and particular types of channels are associated withparticular conditions. The association of N-type channels in conditionsassociated with neural transmission would indicate that compounds of theinvention which target N-type receptors are most useful in theseconditions. Many of the members of the genus of compounds of formula (1)exhibit high affinity for N-type channels. Thus, as described below,they are screened for their ability to interact with N-type channels asan initial indication of desirable function. It is desirable that thecompounds exhibit IC₅₀ values of <1 μM. The IC₅₀ is the concentrationwhich inhibits 50% of the calcium flux at a particular appliedpotential.

There are two distinguishable types of calcium channel inhibition. Thefirst, designated “open channel blockage,” is conveniently demonstratedwhen displayed calcium channels are maintained at an artificiallynegative resting potential of about −100 mV (as distinguished from thetypical endogenous resting maintained potential of about −70 mV). Whenthe displayed channels are abruptly depolarized under these conditions,calcium ions are caused to flow through the channel and exhibit a peakcurrent flow which then decays. Open channel blocking inhibitorsdiminish the current exhibited at the peak flow and can also acceleratethe rate of current decay.

This type of inhibition is distinguished from a second type of block,referred to herein as “inactivation inhibition.” When maintained at lessnegative resting potentials, such as the physiologically importantpotential of −70 mV, a certain percentage of the channels may undergoconformational change, rendering them incapable of being activated—i.e.,opened—by the abrupt depolarization. Thus, the peak current due tocalcium ion flow will be diminished not because the open channel isblocked, but because some of the channels are unavailable for opening(inactivated). “Inactivation” type inhibitors increase the percentage ofreceptors that are in an inactivated state.

In order to be maximally useful in treatment, it is also helpful toassess the side reactions which might occur. Thus, in addition to beingable to modulate a particular calcium channel, it is desirable that thecompound has very low activity with respect to the HERG K⁺ channel whichis expressed in the heart. Compounds that block this channel with highpotency may cause reactions which are fatal. Thus, for a compound thatmodulates the calcium channel, it should also be shown that the HERG K⁺channel is not inhibited. Similarly, it would be undesirable for thecompound to inhibit cytochrome p450 since this enzyme is required fordrug detoxification. Finally, the compound will be evaluated for calciumion channel type specificity by comparing its activity among the varioustypes of calcium channels, and specificity for one particular channeltype is preferred. The compounds which progress through these testssuccessfully are then examined in animal models as actual drugcandidates.

SYNTHESIS OF THE INVENTION COMPOUNDS

The compounds of the invention modulate the activity of calciumchannels; in general, said modulation is the inhibition of the abilityof the channel to transport calcium. As described below, the effect of aparticular compound on calcium channel activity can readily beascertained in a routine assay whereby the conditions are arranged sothat the channel is activated, and the effect of the compound on thisactivation (either positive or negative) is assessed. Typical assays aredescribed hereinbelow.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be acid addition salts involving inorganic or organic acids or thesalts may, in the case of acidic forms of the compounds of the inventionbe prepared from inorganic or organic bases. Suitable pharmaceuticallyacceptable acids and bases are well-known in the art, such ashydrochloric, sulphuric, citric, acidic, or tartaric acids and potassiumhydroxide, sodium hydroxide, ammonium hydroxide, caffeine, variousamines, and the like. Methods for preparation of the appropriate saltsare well-established in the art.

In addition, in some cases, the compounds of the invention contain oneor more chiral centers; this is particularly the case where only asingle ring A, B, C or D is substituted. The invention includes theisolated stereoisomeric forms as well as mixtures of stereoisomers invarying degrees of chiral purity.

The compounds of the invention may be synthesized using conventionalmethods. Illustrative of such methods are Schemes 1-3:

Reaction Scheme 1 was used to prepare compounds of the invention withsubstituents in ring A and/or B, where the substituent does not bridgethese rings. This scheme may also be used to prepare compounds withsubstituents in rings C and D by modifying the benzhydril carboxylicacid in the last step of the synthesis. Thus, the method set forth inReaction Scheme 1 was used to synthesize compounds 1-4, 6, 7, 9-15,17-26, 29, 30, 35 and 36.

Similarly, when substituents are present in ring C and/or D, a compoundof formula 4 (but where R² and R¹ are replaced by R⁴ and R⁵) isconverted to a compound of formula 6 by reaction with acetic acidcontaining a leaving group on the α carbon.

For those compounds where a bridge is formed between rings A and B,Reaction Scheme 2 is employed. Thus, this Reaction Scheme was used tosynthesize compounds P16, P27, P28, P31 and P32.

-   -   wherein R^(A) is benzhydril. In instances where rings C and D        are bridged, R^(A) is a benzhydril derivative containing the “X”        bridge.

In all of these cases, the bridged benzhydrils may be furthersubstituted by R¹-R² or R⁴-R⁵ as set forth hereinabove.

For synthesis of compounds with substituents in the piperazine ring,Reaction Scheme 3 is employed. Thus, Reaction Scheme 3 was used tosynthesize compounds P5 and P8.

-   -   wherein R^(A), as defined above, is benzhydril.

For synthesis of compounds where the substituent on the piperazine ringis a keto group, Reaction Scheme 4 is employed.

Compound p34 was synthesized using Reaction Scheme 4.

Preferred Embodiments

The compounds of formula (1) are defined as shown in terms of theembodiments of their various substituents. The substituents may includeoptionally substituted alkyl, aryl, alkaryl and the like.

As used herein, the terms “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent substituents,containing only C and H when they are unsubstituted. Examples includemethyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl,3-butynyl, and the like. Typically, the alkyl, alkenyl and alkynylsubstituents contain 1-10C (alkyl) or 2-10C (alkenyl or alkynyl).Preferably they contain 1-6C (lower alkyl) or 2-6C (lower alkenyl orlower alkynyl).

Heteroalkyl, heteroalkenyl and heteroalkynyl are similarly defined butmay contain 1 or more O, S or N heteroatoms or combinations thereofwithin the backbone residue.

“Acyl” encompasses the definitions of alkyl, alkenyl, alkynyl, each ofwhich is coupled to an additional residue through a carbonyl group,heteroacyl includes the related heteroforms.

“Aromatic” moiety or “aryl” moiety refers to a monocyclic or fusedbicyclic moiety such as phenyl or naphthyl; “heteroaromatic” also refersto monocyclic or fused bicyclic ring systems containing one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits inclusion of 5-membered rings as well as 6-membered rings. Thus,typical aromatic/heteroaromatic systems include pyridyl, pyrimidyl,indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl,benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl,oxazolyl, imidazolyl and the like. Because tautomers are theoreticallypossible, phthalimido is also considered aromatic. Any monocyclic orfused ring bicyclic system which has the characteristics of aromaticityin terms of electron distribution throughout the ring system is includedin this definition. Typically, the ring systems contain 5-12 ring memberatoms.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aromatic andheteroaromatic systems which are coupled to another residue through acarbon chain, including substituted or unsubstituted, saturated orunsaturated, carbon chains, typically of 1-8C, or the hetero formsthereof. These carbon chains may also include a carbonyl group, thusmaking them able to provide substituents as an acyl or heteroacylmoiety.

In general, any alkyl, alkenyl, alkynyl, acyl, or aryl group containedin a substituent may itself optionally be substituted by additionalsubstituents. The nature of these substituents is similar to thoserecited with regard to the primary substituents themselves. Thus, wherean embodiment of a substituent is alkyl, this alkyl may optionally besubstituted by the remaining substituents listed as substituents wherethis makes chemical sense, and where this does not undermine the sizelimit of alkyl per se; e.g., alkyl substituted by alkyl or by alkenylwould simply extend the upper limit of carbon atoms for theseembodiments. However, alkyl substituted by aryl, amino, alkoxy, and thelike would be included. The features of the invention compounds aredefined by formula (1) and the nature of the substituents is lessimportant as long as the substituents do not interfere with the statedbiological activity of this basic structure.

Non-interfering substituents on Ar¹ or Ar², include, but are not limitedto, alkyl, alkenyl, alkynyl, halo, OR, NR₂, SR, —SOR, —SO₂R, —OCOR,—NRCOR, —NRCONR₂, —NRCOOR, —OCONR₂, —RCO, —COOR, SO₂R, NRSOR, NRSO₂R,—SO₃R, —CONR₂, SO₂NR2, wherein each R is independently H or alkyl(1-8C), —CN, —CF₃, and NO₂, and like substituents. R³ and R⁴ can also beH. Preferred embodiments for R³ and R⁴ are H, alkyl (1-10C) or aheteroatom-containing form thereof, each optionally substituted,especially (1-4C) alkyl; alkoxy (1-8C), acylamido, aryloxy,arylalkyloxy, especially wherein the aryl group is a phthalimido group,and alkyl or arylalkyl amine.

Particularly preferred embodiments of the invention are those whereinonly one or two of the rings are substituted and wherein the number ofsubstituents on a single ring is three or less. Particularly preferredsubstituents for rings A and/or B include halo, especially chloro; CF₃;optionally substituted, optionally heteroatom-containing alkyl, alkenyl,aryl, alkyl aryl, alkenyl aryl, phenoxy, and the like. Where thesubstituents on these moieties contain alkyl or aryl groups, these alsomay optionally be substituted. Also preferred are bridging substituentscontaining heteroatoms. The bridge between rings A and B or C and Dpreferably contain 1-3 members including preferably (CR₂)_(m) where m is1-3; (CR₂)₁ NR(CR₂)₁ (CR₂)₁ O(CR₂)₁ (CR₂)₁ S(CR₂)₁ where S is optionallyoxidized, CR₂, O, NR, and optionally oxidized S.

Particularly preferred substituents for the piperazine ring includeCOOR, especially COOH and COOEt, alkyl, and alkenyl, (as defined aboveand optionally containing heteroatoms and all optionally substituted)and halo.

Preferred substituents for rings C and D are similar to those for A andB. Also preferred are compounds of formula (1) wherein all of n¹-n⁵ are0.

Libraries and Screening

The compounds of the invention can be synthesized individually usingmethods known in the art per se, or as members of a combinatoriallibrary.

Synthesis of combinatorial libraries is now commonplace in the art.Suitable descriptions of such syntheses are found, for example, inWentworth, Jr., P., et al., Current Opinion in Biol. (1993) 9:109-115;Salemme, F. R., et al., Structure (1997) 5:319-324. The librariescontain compounds with various substituents and various degrees ofunsaturation, as well as different chain lengths. The libraries, whichcontain, as few as 10, but typically several hundred members to severalthousand members, may then be screened for compounds which areparticularly effective against a specific subtype of calcium channel,i.e., the N-type channel. In addition, using standard screeningprotocols, the libraries may be screened for compounds which blockadditional channels or receptors such as sodium channels, potassiumchannels and the like.

Methods of performing these screening functions are well known in theart. These methods can also be used for individually ascertaining theability of a compound to agonize or antagonize the channel. Typically,the channel to be targeted is expressed at the surface of a recombinanthost cell such as human embryonic kidney cells. The ability of themembers of the library to bind the channel to be tested is measured, forexample, by the ability of the compound in the library to displace alabeled binding ligand such as the ligand normally associated with thechannel or an antibody to the channel. More typically, ability toantagonize the channel is measured in the presence of calcium ion andthe ability of the compound to interfere with the signal generated ismeasured using standard techniques. In more detail, one method involvesthe binding of radiolabeled agents that interact with the calciumchannel and subsequent analysis of equilibrium binding measurementsincluding, but not limited to, on rates, off rates, K_(d) values andcompetitive binding by other molecules.

Another method involves the screening for the effects of compounds byelectrophysiological assay whereby individual cells are impaled with amicroelectrode and currents through the calcium channel are recordedbefore and after application of the compound of interest.

Another method, high-throughput spectrophotometric assay, utilizesloading of the cell lines with a fluorescent dye sensitive tointracellular calcium concentration and subsequent examination of theeffects of compounds on the ability of depolarization by potassiumchloride or other means to alter intracellular calcium levels.

As described above, a more definitive assay can be used to distinguishinhibitors of calcium flow which operate as open channel blockers, asopposed to those that operate by promoting inactivation of the channel.The methods to distinguish these types of inhibition are moreparticularly described in the examples below. In general, open-channelblockers are assessed by measuring the level of peak current whendepolarization is imposed on a background resting potential of about−100 mV in the presence and absence of the candidate compound.Successful open-channel blockers will reduce the peak current observedand may accelerate the decay of this current. Compounds that areinactivated channel blockers are generally determined by their abilityto shift the voltage dependence of inactivation towards more negativepotentials. This is also reflected in their ability to reduce peakcurrents at more depolarized holding potentials (e.g., −70 mV) and athigher frequencies of stimulation, e.g., 0.2 Hz vs. 0.03 Hz.

Utility and Administration

For use as treatment of human and animal subjects, the compounds of theinvention can be formulated as pharmaceutical or veterinarycompositions. Depending on the subject to be treated, the mode ofadministration, and the type of treatment desired—e.g., prevention,prophylaxis, therapy; the compounds are formulated in ways consonantwith these parameters. A summary of such techniques is found inRemington's Pharmaceutical Sciences, latest edition, Mack PublishingCo., Easton, Pa., incorporated herein by reference.

In general, for use in treatment, the compounds of formula (1) may beused alone, as mixtures of two or more compounds of formula (1) or incombination with other pharmaceuticals. Depending on the mode ofadministration, the compounds will be formulated into suitablecompositions to permit facile delivery.

Formulations may be prepared in a manner suitable for systemicadministration or topical or local administration. Systemic formulationsinclude those designed for injection (e.g., intramuscular, intravenousor subcutaneous injection) or may be prepared for transdermal,transmucosal, or oral administration. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers,preservatives and the like. The compounds can be administered also inliposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms asliquid solutions or suspensions or as solid forms suitable for solutionor suspension in liquid prior to injection or as emulsions. Suitableexcipients include, for example, water, saline, dextrose, glycerol andthe like. Such compositions may also contain amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like, such as, for example, sodium acetate, sorbitanmonolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See,for example, U.S. Pat. No. 5,624,677.

Systemic administration may also include relatively noninvasive methodssuch as the use of suppositories, transdermal patches, transmucosaldelivery and intranasal administration. Oral administration is alsosuitable for compounds of the invention. Suitable forms include syrups,capsules, tablets, as in understood in the art.

For administration to animal or human subjects, the dosage of thecompounds of the invention is typically 0.1-15 mg/kg, preferably 0.1-1mg/kg. However, dosage levels are highly dependent on the nature of thecondition, the condition of the patient, the judgment of thepractitioner, and the frequency and mode of administration.

The following examples are intended to illustrate but not to limit theinvention.

Preparation 1 General Procedure for Preparation of Compounds of Formula(1) from Benzhydrilpiperazine Derivatives

N-(Diphenylmethyl)piperazine (0.5 mmole) is dissolved in dry THF (10ml). To each reaction flask is added powdered K₂CO₃ and acid chloride ofthe formula Φ₂CHCH₂—CO—Cl (0.7 mmole), wherein one phenyl group issubstituted. The reaction is stirred at RT for 2h and quenched with 105NaOH (10 ml) and extracted with EtOAc (10 ml). The organic layer iswashed with 10% NaOH (4×) and dried over sodium sulfate, concentrated,and purified by column chromatography (silica gel, 1:1 hex:EtOAc) togive the desired amide.

Preparation 2 Model for Synthesis of Substituted1-(4-Benzhydryl-piperazin-1-yl)-3,3-diphenyl-propan-1-one

The model is conducted synthesizing the unsubstituted form.A. Synthesis of Diphenyl-methanol

A solution of benzaldehyde (7.34 mmol) in dry ether (10 ml) was addedslowly to a solution of phenyhnagnesium bromide (2.3 ml, 6.98 mmol, 3.0M in ether) under nitrogen. The mixture was heated to reflux for 1 hourthen cooled to 0° C. and hydrolysed with 1 N HCl (40 ml). The aqueousphase was extracted with ether (3×) and combined organic layer driedover MgSO₄. The crude product was purified using hexane: ethyl acetate(5:1) as eluant to give 1.5 g of pure product.B. Synthesis of Cholorodiphenylmethane

To a solution of diphenyl carbinol (11.06 mmol) in dry benzene (20 ml)was added SOCl₂ (8.25 ml, 110 mmol) and anhydrous CaCl₂ (2 g). Themixture was heated under reflux for 2 hours and then cooled and stirredat room temperature overnight. It was then filtered and solvent removedin vacuo to give a pale yellow oil and was used in the next step withoutfurther purification.C. Synthesis of 1-Benzhydryl-piperazine

A mixture of cholorodiphenylmethane (17.4 mmol) in butanone (20 ml),anhydrous piperazine (5.98 g, 69.6 mmol), anhydrous K₂CO₃ (2.40 g, 17.4mmol) and KI (2.88 g, 17.4 mmol) was refluxed under nitrogen for 18hours. The mixture was then cooled and filtered and the solvent removedin vacuo. The residue was dissolved in CH₂Cl₂ (100 ml) and washed withwater (30 ml). Drying and removal of the solvent followed bychromatography (CH₂Cl₂:CH₃OH:NH₄OH 90:10:0.5) afforded desired productin 57% yield.D. Synthesis of1-(4-Benzhydryl-piperazin-1-yl)-3,3-diphenyl-propan-1-one

To a solution of 1-Benzhydryl-piperazine (2.08 mmol) in dry CH₂Cl₂ (40ml) was added 3,3-diphenylpropanoic acid (0.472 g, 2.08 mmol) undernitrogen. To the reaction was added EDC (0.797 g, 4.16 mmol) and DMAP(cat) and the reaction mixture stirred under nitrogen at roomtemperature overnight. The reaction was then concentrated under reducedpressure. The residue dissolved in ethyl acetate: water (10:1) (150 ml).The organic was washed with water (30 ml, 2×) and 10% NaOH (30 ml) anddried over MgSO₄ and evaporated to dryness. The resulting residue waspurified by column chromatography using hexane: ethyl acetate (3:1) togive title compound in 78% yield.

In the foregoing procedure, substituted forms of the reagents—as notedby “R” —are employed.

EXAMPLE 1 Synthesis of1-{4-[(4-Chloro-phenyl)-phenyl-methyl]-piperazin-1-yl}-3,3-diphenyl-propan-1-one

A. Synthesis of (4-Chloro-phenyl)-phenyl-methanol

A solution of 4-chlorobenzaldehyde (1.03 g, 7.34 mmol) in dry ether (10ml) was added slowly to a solution of phenylmagnesium bromide (2.3 ml,6.98 mmol, 3.0 M in ether) under nitrogen. The mixture was heated toreflux for 1 hour then cooled to 0° C. and hydrolysed with 1 N HCl (40ml). The aqueous phase was extracted with ether (3×) and combinedorganic layer dried over MgSO₄. The crude product was purified usinghexane: ethyl acetate (5:1) as eluant to give 1.5 g of pure product.B. Synthesis of 1 -Chloro-4-(chloro-phenyl-methyl)-benzene

To a solution of (4-chloro-phenyl)-phenyl-methanol (2.41 g, 11.06 mmol)in dry benzene (20 ml) was added SOCl₂ (8.25 ml, 110 mmol) and anhydrousCaCl₂ (2 g). The mixture was heated under reflux for 2 hours and thencooled and stirred at r.t. overnight. It was then filtered and solventremoved in vacuo to give a pale yellow oil and was used in the next stepwithout further purification.C. Synthesis of 1-[(4-Chloro-phenyl)-phenyl-methyl]-piperazine

A mixture of 1-chloro-4-(chloro-phenyl-methyl)-benzene (4.12 g, 17.4mmol) in butanone (20 ml), anhydrous piperazine (5.98 g, 69.6 mmol),anhydrous K₂CO₃ (2.40 g, 17.4 mmol) and KI (2.88 g, 17.4 mmol) wasrefluxed under nitrogen for 18 hours. The mixture was then cooled andfiltered and the solvent removed in vacuo. The residue was dissolved inCH₂Cl₂ (100 ml) and washed with water (30 ml). Drying and removal of thesolvent followed by chromatography (CH₂Cl₂:CH₃OH:NH₄OH 90:10:0.5)afforded desired product in 57% yield.D. Synthesis of1-{4-[(4-Chloro-phenyl)-phenyl-methyl]-piperazin-1-yl}-3,3-diphenyl-propan-1-one

To a solution of 1-[(4-chloro-phenyl)-phenyl-methyl]-piperazine (0.59 g,2.08 mmol) in dry CH₂Cl₂ (40 ml) was added 3,3-diphenylpropanoic acid(0.472 g, 2.08 mmol) under nitrogen. To the reaction was added EDC(0.797 g, 4.16 mmol) and DMAP (cat) and the reaction mixture stirredunder nitrogen at room temp. over night. The reaction was thenconcentrated under reduced pressure. The residue dissolved in ethylacetate: water (10:1) (150 ml). The organic was washed with water (30ml, 2×) and 10% NaOH (30 ml) and dried over MgSO₄ and evaporated todryness. The resulting residue was purified by column chromatographyusing hexane: ethyl acetate (3:1) to give desired product in 78% yield.

EXAMPLE 2 Synthesis of3,3-Diphenyl-1-[4-(9H-thioxanthen-9-yl)-piperazin-1-yl]-propan-1-one

A. Synthesis of 9H-Thioxanthen-9-ol

Xanthone (2.1 g, 9.9 mmols) was reduced with an excess of sodiumborohydride (5.0 g, 0.13 mol) in 95% EtOH (50 mL). After stirring for 45min., 10 mL of water was added and the mixture warmed on a steam bath.Addition of ice caused the precipitation of thioxanthen-9-ol, which wasthen washed with water and dried. Yield 2.0 g, mp. 102-105° C.B. Synthesis of3,3-Diphenyl-1-[4-(9H-thioxanthen-9-yl)-piperazin-1-yl]-propan-1-one

The thioxanthen-9-ol (1.0 g, 4.66 mmol) was dissolved in dry CH₂Cl₂ (25mL) and 3.0 mL of 2-6-Lutidine and cooled in an ice-H₂O bath. Triflicanhydride (0.87 mL, 5.12 mmol) was added via syringe, and the resultingred reaction mixture was stirred at 0° C. After 30 min, compound 9(3,3-diphenyl-1-piperazin-1-yl-propan-1-one) (1.64 g, 5.6 mmol) wasadded at 0° C. and stirred at this temperature for 1 hour. The reactionmixture was then stirred at room temperature overnight. The mixture wasquenched with water, and the organic phase washed with water, saturatedNaCl, dried over MgSO₄, and evaporated. The crude product was purifiedby column chromatography on silica (Hexane:EtOAc 1:1) to give 0.75 gpure product.

EXAMPLE 3 Synthesis of4-Benzhydryl-1-(3,3-diphenyl-propionyl)-piperazine-2-carboxylic acidethyl ester

A. Synthesis of Piperazine 2-carboxylic acid ethyl ester

Compound 15 (1 eq.) was dissolved, with warming, in EtOH, andhydrogenated over 10% Pd-C at room temperature and atmospheric pressureuntil H₂ uptake ceased. The mixture was filtered through Celite and thesolvent evaporated, giving an oil which was distilled under reducedpressure.B. Synthesis of 4-Benzhydryl-piperazine-2-carboxylic acid ethyl ester

A mixture of piperazine 2-carboxylic acid ethyl ester 16 (1.0 g, 6.32mmol), bromodiphenylmethane 17 (1.56 g, 6.32 mmol), K₂CO₃ (1.05 g, 7.58mmol) in anhydrous DMF (20 ml) was stirred at room temperature for threedays. The mixture was then diluted with EtOAc (100 ml), washed withwater (2×30 ml), brine (2×30 ml), dried over MgSO₄ and evaporated.Purification by column chromatography using CH₂Cl₂:CH₃OH (15:1) gaveproduct in 75% yield.C. Synthesis of4-Benzhydryl-1-(3,3-diphenyl-propionyl)-piperazine-2-carboxylic acidethyl ester

To a solution of 4-Benzhydryl-piperazine-2-carboxylic acid ethyl ester(0.5 g, 1.54 mmol) in dry CH₂Cl₂ (25 ml) was added 3,3-diphenylpropanoicacid (0.35 g, 1.54 mmol) under nitrogen. To the reaction was added EDC(0.59 g, 3.08 mmol) and DMAP (cat) and the reaction mixture stirredunder nitrogen at room temperature overnight. The reaction was thenconcentrated under reduced pressure. The residue dissolved in ethylacetate: water (10:1) (100 ml). The organic was washed with water (20ml, 2×) and 10% NaOH (20 ml) and dried over MgSO₄ and evaporated todryness. The resulting residue was purified by column chromatographyusing hexane: ethyl acetate (3:1) to give title compound in 73% yield.

EXAMPLE 4 Synthesis of4-Benzhydryl-1-(3,3-diphenyl-propionyl)-piperazine-2-carboxylic acid

A mixture of4-Benzhydryl-1-(3,3-diphenyl-propionyl)-piperazine-2-carboxylic acidethyl ester (0.51 g, 0.957 mmol), and LiOH.H₂O (0.12 g, 2.87 mmol) inTHF/MeOH/H₂O (15:5:5) was stirred at room temperature for two days. Thesolvent was evaporated under reduced pressure, the residue was dissolvedin water, acidified with 1N HCl to pH 3. The product was extracted withEtOAc, dried with MgSO₄, and evaporated under reduced pressure. Theproduct was purified by column chromatography (CH₂Cl₂:MeOH 15:1) to givethe title compound in 95% yield.

EXAMPLE 5 Synthesis of1-Benzhydryl-4-(3,3-diphenyl-propionyl)-piperazin-2-one

A. Synthesis of 2-ketopiperazine

A solution of bromoethylacetate (10 g, 59.8 mmol) in absolute ethanol(80 ml) is slowly added at room temperature to a solution ofethylenediamine (36 g, 598 mmol) in absolute ethanol (140 ml). Theaddition requires about three hours and the mixture is allowed to standfor an additional two hours. Sodium ethoxide (21% wt, 22 ml, 59.8 mmol)was added dropwise. The mixture was stirred at room temperatureovernight and solvent was then evaporated. DMF (40 ml) was added toresidue and stirred at 60-70° C. for 24 hours. The salt was filtered andthe solvent was evaporated. The residue was purified by columnchromatography using CH₂Cl₂:MeOH:NH₄OH (90:10:0.1) to give a yellowsolid in 45% yield.B. Synthesis of 4-(3,3-diphenyl-propionyl)-piperazin-2-one

To a solution of 2-ketopiperazine (0.7 g, 7.0 mmol) in dry CH₂Cl₂ (30ml) was added 3,3-diphenylpropanoic acid (1.9 g, 8.4 mmol) undernitrogen. To the reaction was added EDC (1.7 g, 9.1 mmol) and DMAP (cat)and the reaction mixture stirred under nitrogen at room temperatureovernight. The reaction was then concentrated under reduced pressure.The residue dissolved in ethyl acetate: water (10:1) (100 ml). Theorganic was washed with water (20 ml, 2×) and 10% NaOH (20 ml) and driedover MgSO₄ and evaporated to dryness. The resulting residue was purifiedby column chromatography using hexane: ethyl acetate (2:1) to giveproduct in 70% yield.C. Synthesis of 1-Benzhydryl-4-(3,3-diphenyl-propionyl)-piperazin-2-one

To a solution of 4-(3,3-diphenyl-propionyl)-piperazin-2-one (0.5 g, 1.62mmol) in dry DMF (15 ml) was added NaH (60%, 75 mg, 1.86 mmol) andresulting mixture stirred for half an hour. To this mixturebromodiphenylmethane (0.40 g, 1.62 mmol) was added and the mixturestirred at 100° C. over night. It was then cooled, EtOAc (100 ml) wasadded and washed with water (2×), brine (1×). The organic phase was thendried and evaporated to give a residue which upon column chromatographyusing CH₂Cl₂:MeOH (20:1) gave the title compound in 65% yield.

EXAMPLE 6 Assessment of Calcium Channel Blocking Activity

Antagonist activity was measured using whole cell patch recordings onhuman embryonic kidney cells either stably or transiently expressing ratα_(1B)+α_(2b)+β_(1b) channels (N-type channels) with 5 mM barium as acharge carrier.

For transient expression, host cells, such as human embryonic kidneycells, HEK 293 (ATCC# CRL 1573) were grown in standard DMEM mediumsupplemented with 2 mM glutamine and 10% fetal bovine serum. HEK 293cells were transfected by a standard calcium-phosphate-DNAcoprecipitation method using the rat α_(1B)+β_(1b)+α₂δ N-type calciumchannel subunits in a vertebrate expression vector (for example, seeCurrent Protocols in Molecular Biology).

After an incubation period of from 24 to 72 hrs the culture medium wasremoved and replaced with external recording solution (see below). Wholecell patch clamp experiments were performed using an Axopatch 200Bamplifier (Axon Instruments, Burlingame, Calif.) linked to an IBMcompatible personal computer equipped with pCLAMP software. Borosilicateglass patch pipettes (Sutter Instrument Co., Novato, Calif.) werepolished (Microforge, Narishige, Japan) to a resistance of about 4 MΩwhen filled with cesium methanesulfonate internal solution (compositionin MM: 109 CsCH₃SO₄, 4 MgCl₂, 9 EGTA, 9 HEPES, pH 7.2). Cells werebathed in 5 mM Ba⁺⁺ (in mM: 5 BaCl₂, 1 MgCl₂, 10 HEPES, 40tetraethylammonium chloride, 10 glucose, 87.5 CsCl pH 7.2). Current datashown were elicited by a train of 100 ms test pulses at 0.066 Hz from−100 mV and/or −80 mV to various potentials (min. −20 mV, max. +30 mV).Drugs were perfused directly into the vicinity of the cells using amicroperfusion system.

Normalized dose-response curves were fit (Sigmaplot 4.0, SPSS Inc.,Chicago, Ill.) by the Hill equation to determine IC₅₀ values.Steady-state inactivation curves were plotted as the normalized testpulse amplitude following 5 s inactivating prepulses at +10 mVincrements. Inactivation curves were fit (Sigmaplot 4.0) with theBoltzman equation, I_(peak) (normalized)=1/(1+exp((V−V_(h))z/25.6)),where V and V_(h) are the conditioning and half inactivation potentials,respectively, and z is the slope factor.

Using the procedure set forth above, various compounds of the inventionwere tested for their ability to block N-type calcium channels. Theresults show IC₅₀ values in the range of 0.05≧1 μM, as shown in Table 1.

TABLE 1 Block of α1B N-type Channels 0.067 Hz 0.2 Hz Compound IC₅₀ (μM)IC₅₀ (μM) P1  0.100 0.074 P2  0.200 0.105 P3  0.291 0.111 P4  0.2130.114 P5  0.160 0.120 P6  0.170 0.120 P7  0.213 0.137 P8  0.230 0.140P9  0.230 0.170 P10 (HCl) 0.300 0.190 P10 0.550 0.450 P11 0.370 0.190P12 0.340 0.190 P13 0.300 0.210 P14 0.320 0.210 P15 0.348 0.217 P160.290 0.220 P17 0.286 0.233 P18 0.324 0.237 P19 0.360 0.249 P20 0.3200.250 P21 0.437 0.252 P22 0.538 0.301 P23 0.490 0.310 P24 0.600 0.380P25 1.090 0.513 P26 0.710 0.533 P27 0.854 0.552 P29 (HCl) >1 >1P29 >1 >1 P30 >1 >1 P32 (HCl) >1 0.830 P34 0.737 0.680

As shown in Table 1, the nature of the substituent has an influence onthe IC₅₀ value.

EXAMPLE 7 Additional Methods

The methods of Example 6 were followed with slight modifications as willbe apparent from the description below.

A. Transformation of HEK Cells:

N-type calcium channel blocking activity was assayed in human embryonickidney cells, HEK 293, stably transfected with the rat brain N-typecalcium channel subunits (α_(1B)+α_(2δ)+β_(1b) cDNA subunits).Alternatively, N-type calcium channels (α_(1B)+α_(2δ)+β_(1b) cDNAsubunits), L-type channels (α_(1C)+α_(2δ)+β_(1b) cDNA subunits) andP/Q-type channels (α_(1A)+α_(2δ)+β_(1b) cDNA subunits) were transientlyexpressed in HEK 293 cells. Briefly, cells were cultured in Dulbecco'smodified eagle medium (DMEM) supplemented with 10% fetal bovine serum,200 U/ml penicillin and 0.2 mg/ml streptomycin at 37° C. with 5% CO₂. At85% confluency cells were split with 0.25% trypsin/1 mM EDTA and platedat 10% confluency on glass coverslips. At 12 hours the medium wasreplaced and the cells transiently transfected using a standard calciumphosphate protocol and the appropriate calcium channel cDNAs. Fresh DMEMwas supplied and the cells transferred to 28° C./5% CO₂. Cells wereincubated for 1 to 2 days to whole cell recording.

B. Measurement of Inhibition:

Whole cell patch clamp experiments were performed using an Axopatch 200Bamplifier (Axon Instruments, Burlingame; Calif.) linked to a personalcomputer equipped with pCLAMP software. The external and internalrecording solutions contained, respectively, 5 mM BaCl₂, 1 mM MgCl₂, 10mM HEPES, 40 mM TEACl, 10 mM glucose, 87.5 mM CsCl (pH 7.2) and 108 mMCsMS, 4 mM MgCl₂, 9 mM EGTA, 9 mM HEPES (pH 7.2). Currents weretypically elicited from a holding potential of −80 mV to +10 mV usingClampex software (Axon Instruments). Typically, currents were firstelicited with low frequency stimulation (0.03 Hz) and allowed tostabilize prior to application of the compounds. The compounds were thenapplied during the low frequency pulse trains for two to three minutesto assess tonic block, and subsequently the pulse frequency wasincreased to 0.2 Hz to assess frequency dependent block. Data wereanalyzed using Clampfit (Axon Instruments) and SigmaPlot 4.0 (JandelScientific).

Table 2 shows the results obtained with several compounds of theinvention which are selective for N-type channels.

TABLE 2 Selectivity of Compounds for N-type Ca²⁺ Channels Tested at 0.1Hz, 5 mM Ba²⁺ N-type P/Q-type IC₅₀ IC₅₀ L-type IC₅₀ P/Q:N Compound (μM)(μM) (μM) ratio L:N ratio P1 0.19 0.97 19.6 5.1:1  103:1 P3 0.1857.59 >>10 41:1 >>54:1 P4 0.251 >>10 >>10 >>40:1  >>40:1 P5 0.073 5.0 21069:1 2877:1 P6 0.16 4.5 133 28:1  831:1 P8 0.36 3.4 37.1 9.4:1   103:1

The results shown in Table 2 are shown graphically in FIGS. 2-5. As wasthe case for IC₅₀ values, specificity for a particular type of channelis dependent on the nature of the substituents.

EXAMPLE 8 Block of α_(1G) T-type Channels

Standard patch-clamp techniques were employed to identify blockers ofT-type currents. Briefly, previously described HEK cell lines stablyexpressing human α_(1G) subunits were used for all the recordings(passage #: 4-20, 37° C., 5% CO₂). To obtain T-type currents, plasticdishes containing semi-confluent cells were positioned on the stage of aZEISS AXIOVERT S100 microscope after replacing the culture medium withexternal solution (see below). Whole-cell patches were obtained usingpipettes (borosilicate glass with filament, O.D.: 1.5 mm, I.D.: 0.86 mm,10 cm length), fabricated on a SUTTER P-97 puller with resistance valuesof ˜5 MΩ (see below for internal medium).

TABLE 3 External Solution 500 ml - pH 7.4, 265.5 mOsm Salt Final mMStock M Final ml CsCl 132 1 66 CaCl₂ 2 1 1 MgCl₂ 1 1 0.5 HEPES 10 0.5 10glucose 10 — 0.9 grams

TABLE 4 Internal Solution 50 ml - pH 7.3 with CsOH, 270 mOsm Salt FinalmM Stock M Final ml Cs-Methanesulfonate 108 — 1.231 gr/50 ml MgCl₂ 2 10.1 HEPES 10 0.5 1 EGTA-Cs 11 0.25 2.2 ATP 2 0.2 0.025 (1 aliquot/2.5ml) T-type currents were reliably obtained by using two voltageprotocols: (1) “non-inactivating”, and (2) “inactivation”

In the non-inactivating protocol, the holding potential is set at −110mV and with a pre-pulse at −100 mV for 1 second prior to the test pulseat −40 mV for 50 ms; in the inactivation protocol, the pre-pulse is atapproximately −85 mV for 1 second, which inactivates about 15% of theT-type channels, as shown below.

Test compounds were dissolved in external solution, 0.1-0.01% DMSO.After ˜10 min rest, they were applied by gravity close to the cell usinga WPI microfil tubing. The “non-inactivated” pre-pulse was used toexamine the resting block of a compound. The “inactivated” protocol wasemployed to study voltage-dependent block, but the initial data shownbelow were mainly obtained using the non-inactivated protocol only. IC₅₀values are shown for various compounds of the invention in Table 5.

TABLE 5 Block of α_(1G) T-type Channels 100 mV 80 mV Compound IC₅₀ (μM)IC₅₀ (μM) P6  0.081 — P9  >1 — P13 <1 — P15 0.063 — P17 No effect — P180.035 — P19 0.745 — P29 0.033 0.004 P30 >1 — P31 0.371 — P33 0.404 — P35— 0.141 P36 — 0.055

Again, the substitution pattern has a dramatic impact on the IC₅₀ value.

1. A method to treat a condition selected from the group consisting ofpain, stroke, epilepsy, anxiety and depression in a subject, whichmethod comprises administering to a subject in need of such treatment anamount of a compound of formula (1) effective to treat said conditionwherein said compound of formula (1) is:

or a pharmaceutically acceptable salt thereof wherein each R¹-R⁵ isindependently optionally substituted alkyl (1-10C), alkenyl (2-10C),alkynyl (2-10C), aryl (6-10C), alkylaryl (7-16C) or alkenylaryl (7-16C)each optionally further containing 1-4 heteroatoms (N, O or S) andwherein said optional substituents may include ═O; or each of R¹-R⁵ isindependently halo, NO₂, SO, SO₂, SO₂NH₂, —OH, SH or NH₂, and wherein R³may be keto if n³=1; and wherein two substituents on adjacent positionsof the same ring may form a 3-7 membered saturated or unsaturated ringfused to said substituted ring, said fused ring itself optionallysubstituted and optionally containing one or more heteroatoms (N, S, O);or wherein a combination of R¹ and R² and/or R⁴ and R⁵ may form a bondor a bridge between phenyl groups A and B and/or C and D; and whereineach n¹-n² and n⁴-n⁵ is independently 0-4, and n³ is 1-4; and/or thecompound of formula (1) is in the form of an isolated stereoisomer; orthe compound of formula (1) is P49 or P50 in FIG. (1) or a salt thereof.2. The method of claim 1 wherein each of R¹, R², R⁴ and R⁵ isindependently halo, or is optionally heteroatom containing and/oroptionally substituted alkyl, alkenyl, aryl, alkylaryl, alkenylaryl, orphenoxy.
 3. The method of claim 1 wherein R¹ and R² and/or R⁴ and R⁵form a bridge of 1-3 members.
 4. The method of claim 1 wherein n³ is 1and R³ is COOH or an alkyl ester thereof.
 5. The method of claim 1wherein all of n¹-n² and n⁴-n⁵ are
 0. 6. The method of claim 1 whereinone of n¹-n² and n⁴-n⁵ is 1 and the other n are
 0. 7. The method ofclaim 1 wherein one of n¹-n² and n⁴-n⁵ is 2 and the other n are
 0. 8.The method of claim 1 wherein one of n¹-n² and n⁴-n⁵ is 3 and the othern are
 0. 9. The method of claim 1 which is compound P37-P50 in FIG. 1 ora salt thereof.
 10. The method of claim 1 wherein the condition is pain.11. The method of claim 1 wherein the condition is stroke.
 12. Themethod of claim 1 wherein the condition is epilepsy.
 13. The method ofclaim 1 wherein the condition is anxiety or depression.
 14. The methodof claim 5 wherein the condition is pain.
 15. The method of claim 5wherein the condition is stroke.
 16. The method of claim 5 wherein thecondition is epilepsy.
 17. The method of claim 5 wherein the conditionis anxiety or depression.